Neural stem cells have been isolated from various regions of both rodent and human nervous systems and expanded in vitro as free floating aggregates (U.S. Pat. No. 5,851,832 to Weiss et al.) or as a monolayer attached to a substratum-coated dish (U.S. Pat. No. 5,753,506 to Johe). Neural stem cells are capable of extended self-renewal and have the ability, under appropriate conditions, to generate one or more subtypes of neurons and glia cells in vitro. By virtue of these properties, neural stem cells and their progeny can be applied for pharmaceutical drug discovery and for cell transplantation in neurological disorders.
The fate of neural stem cells can be controlled by a variety of extracellular factors. Growth factors such as basic Fibroblast Growth Factor (bFGF), Epidermal Growth Factor (EGF), Transforming Growth Factor (U.S. Pat. Nos. 5,851,832 and 5,753,506) and Leukemia Growth Factor (LIF; U.S. Pat. No. 6,103,530 to Carpenter) have all been shown to control the in vitro proliferation of neural stem cells. Growth factor expanded stem cells can differentiate into neuron and glia after mitogen withdrawn from the culture medium, but the proportion of neural stem cells that differentiate into neurons is minimal. However, the proportion of neural stem cells differentiating into the neuronal pathway can be increased by exposing the neural stem cells to various other growth factors such as Platelet Derived Growth Factor (PDGF; U.S. Pat. No. 5,753,506); bFGF (U.S. Pat. No. 5,766,948 to Gage and Jasodha); including neurotrophins such as Brain-Derived Growth Factor (BDNF; Shetty and Turner, J Neurobiol. 1998;35:395–425); Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4; Caldwell et al., Nat Biotechnol. 2001;19:475–9); and Notch antagonists (U.S. Pat. No. 6,149,902 to Artavanis-Tsakonas et al.), to increase the quantity of neurons derived from a certain amount of neural stem cells.
For a variety of drugs used to treat psychiatric disorders, accumulating data from experimental studies are now providing novel insights into the mechanism by which the drugs produce their clinical effects. For example, preliminary data has indicated that treatment with the antidepressant drug fluoxetine increases neurogenesis in adult rat hippocampus (Mahlberg, et al., J. Neurosci. 2000;20:9104–10), which may be the reason for the delayed effects of the drug. In addition, numerous studies have shown that retinoic acid has similar differentiation effects in several mammalian systems, including rat, mouse and human embryonic stem cells and human neuroblastoma cells (Inatani et al., Brain Res 2001; 20:217; Guo et al., Electropheresis 2001; 22:3067; Jelitai et al, J Neurobiol 2002; 51:54–65; Lopez-Carballo et al., J Biol. Chem. PubMed ID: 12000752, e-publication ahead of print; Schuldiner et al., Brain Res 2001; 913:201–5; and Freemantle et al., Oncogene 2002; 21:2880–89). This suggests that diverse mammalian stem cells will be similarly affected by a specific compound or drug. However, further experimental data on the cellular mechanisms of action of drugs such as these are still needed, and a possible application of them outside of a clinical setting has, until now, remained unexplored.
The availability of large quantities of neuronal cells such as neurons or neuroblasts is important for the application of such cells both in vivo and in vitro, e.g., in cell transplantation therapy for neurodegenerative diseases and in vitro drug testing in psychiatric disorders. Such quantities can only be obtained by differentiation of cultured neural stem cells into these cell types, which requires large-scale cell culture using a significant amount of various growth factors and/or neurotrophins. Given the sparse availability of neural stem cells, especially human neural stem cells, the cost of growth factor and neurotrophin preparations, and the inability of some factors to cross the blood-brain barrier, there is a need for more efficient and economically viable strategies for differentiating neural stem cells into neuronal cells such as neuroblasts and neurons. This invention addresses these and other needs in the art.